The goal of Project 2 is to understand the molecular mechanisms responsible for longevity and cancer resistance of long-lived rodents. Naked mole rat is the longest-lived rodent species with the maximum lifespan of 32 years. Furthermore, naked mole rats are highly resistant to cancer. Since naked mole rats are related to the short-lived and cancer-prone laboratory rodents, they represent an ideal model for the studies of longevity and cancer resistance. We identified a novel mechanism of cancer-resistance in the naked mole rat termed early contact inhibition (ECl). Naked mole rat cells are hypersensitive to contact inhibition and arrest proliferation at low cell density. ECl is associated with induction of pi6'^'^^ (p16). We found that cells of the naked mole rat, as well as another long-lived rodent the blind mole rat, secrete large amounts of extremely high molecular weight hyaluronan (HMW-HA), which is required for ECl. The properties of HA depend on the polymer length, with longer polymers being more beneflcial. Remarkably, naked mole rat HA is over six times longer than mouse or human HA. We show that HMW-HA protects cells from oxidative stress and rehioval of HA causes naked mole rat cells to form tumors. Based on these findings, we hypothesize that that HMW-HA plays a key role in the naked mole rat longevity and cancer resistance. Our aims are: (1) Determine signaling pathways leading from HA to cell cycle arrest and ECl. We will test the hypothesis that HMW-HA signaling via CD44 receptor activates expression of pi6, and identify intermediate signaling events. In collaboration with Project 4 we will identify genes from HA and pi6 signaling pathways that are activated by HMW-HA and examine their effect on cell cycle arrest in vivo. (2) Identify mechanisms responsible for cytoprotective properties of HMW-HA. We hypothesize that in addition to being an extracellular scavenger of ROS, HMW-HA triggers signaling events that either suppress apoptosis or activate intacellular antioxidant machinery. We will identify the targets of HA signaling, and test whether expression of naked mole rat HAS2 improves oxidative stress resistance in mice. In collaboration with Project 4 we will analyze the stress-signaling genes showing differential expression in the naked mole rat. In collaboration with Project 3 we will determine whether HA has anti-mutagenic effect. (3) Test whether beneficial effects of HMW-HA from the naked mole rat can be transferred to other mammals. We will use two approaches: generate transgenic mice expressing naked mole rat HAS2 or treat mice with hyaluronidase inhibitors. We will test whether these treatments improve mouse cancer and/or stress resistance. RELEVANCE (See instructions): The naked mole rat is the longest living rodent with the maximum lifespan of 32 years. In addition to its longevity, the naked mole rat has an extraordinary resistance to cancer as tumors have never been observed in these rodents. The goal of Project 2 is to understand the mechanisms behind the extraordinary resistance to cancer in the naked mole rat. We expect that investigation of long-lived mammalian species will unravel mechanisms of cancer resistance and longevity that will be applied to extend human lifespan.